Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Viral vectors have the advantage that the heterologous gene is amplified, leading to potentially high levels of expression, but there are constraints on the size of protein that can be expressed before genetic instability becomes a problem. Furthermore, concerns have been expressed about the ability of autonomously viral vectors carrying foreign genes to spread in the environment. In the past few years, the use of viral vectors for the expression of foreign proteins in plants has attracted considerable attention. There are currently two principal means for the expression of such heterologous proteins using viral vectors in plants: transient and stable genetic transformation.
Stable transformation has the advantage that there are few, if any, limitations on the size or complexity of the proteins that can be expressed; however high expression levels can be difficult to achieve routinely. There have been a number of attempts to develop systems that combine the advantages of the transgenic and viral vector approaches. These have generally involved integrating replication-competent cDNA copies of RNA plant viral genomes into genome of a host plant. Virus-specific RNAs transcribed in the nucleus are then amplified by the virus-encoded RNA-dependent RNA polymerase (RdRp). Early attempts to develop this approach using Brome mosaic virus (BMV; Mori et al., 1993) or Potato virus X (PVX; Angell and Baulcombe, 1997) resulted in low levels of protein expression due to replicating viral RNA invoking efficient post-translation gene silencing (PTGS; Kaido et al., 1995; Angell and Baulcombe; 1997; 1999). Attempts to alleviate this problem have involved either the use of an inducible promoter in the case of BMV (Mori et al., 2001) or by introducing a known suppressor of PTGS in the case of PVX (Mallory et al., 2002).
It has recently been shown that transformation of Nicotiana benthamiana with full-length, replication-competent cDNA copies of both genomic RNAs of Cowpea mosaic virus (CPMV; FIG. 1a) results in a productive infection (Liu et al., 2004). This effect was achieved irrespective of whether the two RNAs were introduced simultaneously by co-transformation or by crossing separate RNA-1 and RNA-2 transgenic lines. Furthermore, inoculation of RNA-2 transgenic plants with RNA-1 also gave rise to an infection, though no infection resulted from the reciprocal experiment. This asymmetric complementation between transgene and inoculated genome segment has been attributed to the fact that RNA-1 can act as an amplicon whose effects in inducing PTGS can be counteracted by the presence of an RNA-2-encoded suppressor of silencing which has been identified as the C-terminal region of the Small (S) coat protein (Liu et al., 2004; Cañizares et al., 2004). It has previously been shown that it is possible to insert heterologous sequences into RNA-2 without affecting its ability to be replicated by RNA-1 (Usha et al., 1993, Lomonossoff and Hamilton, 1999; Gopinath et al., 2000).
Although transgenic plants represent promising production systems for long-term and large-scale needs, the time needed to produce expression cassettes containing multiple transgenes, transform plants, and regenerate and multiply transgenic plants imposes a time constraint in the development of a production process.
Transient expression systems allow for the rapid expression of foreign proteins. However, although viral-based transient expression systems have been widely used for the expression of heterologous proteins in plants, limitations of such known systems appear when large amounts of proteins are needed and when producing complex multimeric proteins like monoclonal antibodies. In 1998, Verch et al. (Journal of Immunological Methods 220 (1998) 69-75) report, for the first time, expression of a full size antibody in plants using a viral vector. The paper presents expression of a full size antibody upon co-infection of Nicotiana benthainiana plants with two independent TMV genomic RNAs containing the antibody light and heavy chains, respectively. However, although the authors conclude that assembled antibodies were produced, the level of production was too low to be feasible for commercial antibody production purposes.
Agro-infiltration in detached leaves has also been used for the transient production of antibodies. This transient expression system, first published by Kapila et al. (Plant Science 122 (1997) 101-108) relies on the vacuum-forced entry of Agro-bacteria in leaf tissue, followed by infection of plant cells by the bacteria, transfer of the T-DNA into the nucleus of the plant cells, and transient expression of the gene or genes of interest. The application of Agro-infiltration for full size antibody production has first been reported by Vaquero et al. (PNAS 96 (1999) 11128-11133). In that paper, the authors present the expression of recombinant antibodies specific for the human carcinoembryonic antigen (CEA), a tumor cell surface antigen. Other reports of the use of vacuum-based Agro-infiltration for the production of full size antibodies in plants include Kathuria et al. (Current Science 82 (2002) 1452-1457), Rodriguez et al. (Biotechnology and Bioengineering 89 (2005) 188-194), and Hull et al. (Vaccine 23 (2005) 2082-2086). In opposition to stable transformation of plants, the Agro-infiltration transient expression system is extremely rapid and multiple transgenes can be expressed simultaneously in the same cells simply by co-infiltrating a mixture of Agrobacteria, each strain bearing one gene of interest. The production of antibodies using co-infiltration is exemplified in D'Aoust et al. (Efficient and reliable production of pharmaceuticals in alfalfa, in Molecular Farming, ISBN 3527307869) and Hull et al (Vaccine 23 (2005) 2082-2086).
To date, combining Agro-infiltration and viral vectors into a transient expression system has proven to be very efficient for single protein production. For example, Marillonnet et al. (PNAS 101 (2004) 6852-6857) show extremely high expression levels of GFP in Nicotiana benthamiana upon Agro-inoculation of leaves with Agrobacteria containing a TMV-based viral expression cassette engineered to produce recombinant GFP. However, the co-expression of different sub-units in the same cells using the TMV expression system has not been possible. At the Conference on Plant-Made Pharmaceuticals in Montreal (Jan. 30 to Feb. 2, 2005), the results presented by Yuri Gleba (Icon genetics) showed that upon infection of plants with two different TMV-based viral vectors, competing vectors rapidly segregate and cells accumulated either one or the other RNA of interest, but not both. A solution to this problematic competition between viral vectors was presented at the Plant-Based Vaccines and Antibodies meeting (Jun. 8 to 10, 2005) by Sylvestre Marillonnet (Icon Genetics). The presentation entitled “Expression of protein fusions and of single chain and monoclonal antibodies in plants using viral vectors” proposed a co-infiltration strategy in which elements from different viruses (TMV and PVX) were used when co-expression of different proteins was needed in the same cells. It was shown that, in Nicotiana benthamiana leaves, the TMV- and PVX-based vectors do not compete and that high levels of two proteins in the same cells can be achieved using this strategy. Thus, the production of monoclonal antibodies using this transient expression system combining Agro-inoculation and viral expression vectors is possible. Nonetheless, despite successfully producing antibodies in plants by Agro-inoculation, the combined TMV-PVX viral vector system is dependant on coordinate expression and amplification of independent RNAs from different RdRPs.
In view of the foregoing, it is clear that a need exists for an efficient viral-based transient expression system capable of producing two different proteins in the same cells, particularly in cases high levels of protein production have been difficult to achieve.